Development of an implantable three-dimensional model of a functional pathogenic multispecies biofilm to study infected wounds

Chronic wounds cannot heal due to impairment of regeneration, mainly caused by the persistent infection of multispecies biofilms. Still, the effects of biofilm wound infection and its interaction with the host are not fully described. We aimed to study functional biofilms in physiological conditions in vitro, and their potential effects in health and regeneration in vivo. Therefore, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis were seeded in collagen-based scaffolds for dermal regeneration. After 24 h, scaffolds had bacterial loads depending on the initial inoculum, containing viable biofilms with antibiotic tolerance. Afterwards, scaffolds were implanted onto full skin wounds in mice, together with daily supervision and antibiotic treatment. Although all mice survived their health was affected, displaying fever and weight loss. After ten days, histomorphology of scaffolds showed high heterogeneity in samples and within groups. Wounds were strongly, mildly, or not infected according to colony forming units, and P. aeruginosa had higher identification frequency. Biofilm infection induced leucocyte infiltration and elevated interferon-γ and interleukin-10 in scaffolds, increase of size and weight of spleen and high systemic pro-calcitonin concentrations. This functional and implantable 3D biofilm model allows to study host response during infection, providing a useful tool for infected wounds therapy development.


Results
Formation of biofilms in 3D scaffolds in vitro. As described in the material and method section, a mixture of P. aeruginosa, S. aureus and E. faecalis was incorporated in a 3D collagen-based scaffold that is clinically used for dermal regeneration (Fig. 1). After 24 h of incubation, bacteria remained metabolically active as shown by formation of formazan blue in the MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) assay. Macroscopic visualization of scaffold's front view ( Fig. 2A, upper panel) showed homogeneous distribution of bacteria across the samples seeded with low bacterial loads, whereas in samples with higher loads an increase in the formation of formazan blue was observed in the center of the scaffolds. At both low-and high-density loads, higher magnification showed the presence of bacteria attached to the scaffold fibers, forming a tridimensional structure. Side view images shows a homogeneous vertical distribution at lower bacterial loads, with bacterial aggregates evenly distributed across the scaffolds, while crystals were predominantly concentrated in the upper region of the scaffolds when bacteria were seeded at higher loads ( Fig. 2A, lower panel).
No MTT reduction was observed in control non-seeded scaffolds, whereas a quantitative analysis showed a significant difference among the groups seeded with high and low bacterial loads, which was not directly proportional to the seeded bacterial number (Fig. 2B). CFU counts per scaffold were 10 2 CFU/mL at day 0 ( Fig. 2C) and increased to 10 6 CFU/mL in 24 h, reaching 10 10 CFU/mL in 48 h (Fig. 2B).
To visualize the structure of seeded microorganisms over the biomaterial, scaffolds were analyzed by different microscopic techniques. Confocal laser scanning microscopy (CLSM) analysis showed bacterial aggregates attached to the surface of the scaffold, forming a biofilm-like structure, which was more prominent in the scaffolds seeded with higher bacterial loads (Fig. 3A, upper panels). Z-view shows that bacteria distributed homogeneously in the surface, with the presence of bacterial colonies established in the inner cavities of the material (Fig. 3A, lower panels). Scanning electron microscopy (SEM) revealed a more detailed analysis of the biofilm structure, showing a rather smooth surface in the control non-seeded scaffolds, compared to scaffolds www.nature.com/scientificreports/ aureus and E. faecalis were diluted and seeded on collagen scaffolds to induce biofilm formation. After 24 h of incubation, the biofilm in vitro model was analyzed in means of metabolic activity, bacterial loads, structure and antibiotic tolerance. (B) Once characterized, biofilm-containing scaffolds were implanted in bilateral full-skin defects in mice during 10 days to evaluate the effect over general health parameters through daily supervision. Further analysis of the animal samples were performed, including histology and immunohistochemistry, bacterial loads, pro-calcitonin and cytokine levels. Image was made with BioRender. Figure 2. Viability, distribution, and bacterial loads of biofilms formed in scaffolds. (A) MTT assay of scaffolds seeded with 10 2 or 10 8 cells/mL or without cells (control) and incubated for 24 h showed equal distribution of viable biofilms across the scaffolds in both, front and side views. In higher magnification of scaffolds, bacterial accumulation was observed (arrow heads). (B) Quantitation shows that metabolic activity increased according to the initial cell density. (C) Biofilm-containing scaffolds were diluted and plated in TSS agar for CFU counting. Quantitation shows over 10 5 CFU/scaffold after 18 h incubation of scaffolds, and up to 10 10  www.nature.com/scientificreports/ containing bacteria that fully colonize the surface of the material at higher bacterial loads (Fig. 3B). Additionally, SEM images revealed that most bacteria spotted in biofilm-containing scaffolds are rod-shaped cells embedded in noticeable amounts of extra polymeric substance (EPS).
In vitro functionality of biofilms formed in 3D scaffolds. Once the presence of viable biofilms in 3D scaffolds was confirmed, we decided to study their functionality in terms of antibiotic tolerance, by comparing them to planktonic cultures of the same species. Biofilm-containing scaffolds and bacterial planktonic suspensions were treated with high concentrations of ciprofloxacin or gentamicin, and their antibiotic effect was analyzed (Fig. 4). The microscopic visualization of scaffolds shows a clear reduction of bacterial biofilm biomass after treatment with each antibiotic, where bacteria directly attached to the scaffold preserved a biofilm-like    Fig. S1) showed that a concentration of 10 µg/mL of gentamicin and 1 µg/mL of ciprofloxacin inhibited planktonic bacterial growth and activity, which was confirmed through measurement of OD 600 nm and XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assays. To quantify this effect, an XTT assay of planktonic cultures and biofilm-containing scaffolds treated with antibiotics ( Fig. 4B) showed no metabolic activity for planktonic cells, while biofilms remained viable after treatment with each antibiotic. No statistical differences were found between planktonic and biofilm cells in control media.

Implantation of biofilm-containing scaffolds in vivo.
After characterization of the in vitro 3D biofilm model, an in vivo study was performed. In a pilot experiment, biofilm-containing scaffolds were implanted on bilateral full-thickness skin wounds in mice. Despite adequate analgesia, animal care and cauterization of bleeding vessels during surgery, all mice died from sepsis after one-two days of implantation ( Supplementary Fig. S2).
To prevent this, further in vivo assays included the use of antibiotic and antipyretic treatment. Ciprofloxacin and meloxicam were daily administered to animals (Fig. 5A) resulting in 100% mice survival after biofilm-containing scaffold implantation. During the first two days a few mice showed bleeding around the suture knots, and after ten days there were no signs of bleeding around the scaffolds nor significant wound contraction in either group (Fig. 5B). Implanted sterile scaffolds (control), showed no macroscopic signs of infection or inflammation. In contrast, some animals with biofilm-containing scaffolds showed a yellow secretion in the surrounding wound areas or under the scaffold (Fig. 5B). A daily supervision of animal's health showed a detrimental effect of biofilm implantation over global health parameters (Fig. 5C). Thus, the health score obtained from the daily supervision guide, was significantly increased since day 3 in mice implanted with biofilm-containing scaffolds. A significant difference in body weight was also observed between both groups, as biofilm-implanted mice did not recover their initial weight (Fig. 5D). Finally, during the first four days, the biofilm-implanted group showed a significant increase in the body temperature at the back of mice, which was recovered after five days (Fig. 5E). Relative body weight (% of initial weights at day 0), shows that biofilm-infected group did not recover their weight. (E) Body temperature shows that biofilm-infected group suffers from initial fever but stabilizes temperature at latest days. Scale bar represents 10 mm for B. Values plotted are mean ± SD (N = 8 per group). Two-way ANOVA and Sidak's multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001. www.nature.com/scientificreports/ Systemic and local infection process due to biofilm implantation. Next, scaffolds were removed, and the effect of a possible infection process was studied. Here, paraffin sections of biofilm-containing scaffolds prior to implantation and after ten days in vivo were sectioned and stained for histological analysis (Fig. 6A). Bacterial aggregates were mainly accumulated in the upper region of the biomaterial before implantation, but after ten days such bacterial aggregates were widespread over the scaffold. Sterile implanted scaffolds show cellularization of fibroblast, without leucocyte infiltration, compared to biofilm-implanted scaffolds that showed a considerable amount of polymorphonuclear (PMN) cells, embedded within an abundant biofilm structure. Hematoxylin and eosin (H&E) stained sections of skin surrounding wound area (Fig. 6B) did not show considerable differences due the presence of bacteria, except for an increased presence of leukocyte infiltrate in the adipose tissue of the biofilm model. To discard a systemic infection or sample cross contamination, blood collected from animals was analyzed for CFU counting. Results showed that in both groups no bacterial growth was observed in blood agar plates (Data not shown). To corroborate the presence and distribution of bacterial species seeded in the scaffold, these were identified in situ using specific antibodies (Fig. 6C). Then, CFU/g of scaffolds were quantified (Fig. 6D), comparing scaffolds before implantation (in vitro) and after ten days in vivo. Control scaffolds showed to be sterile, with no bacterial growth, whereas biofilm containing scaffolds in vitro showed mean values of 10 7 CFU/g. Once implanted in full thickness wounds, biofilms reached over 10 9 CFU/g of scaffold despite the ciprofloxacin therapy. To further evaluate the infection process induced by biofilms, pro-calcitonin levels were quantified in serum (Fig. 6E). Although pro-calcitonin basal levels were high for animals implanted with sterile scaffolds (control), biofilm implantation together with antibiotic therapy resulted in significantly increased levels, with over 500 pg/mL of pro-calcitonin (biofilm), while animals that suffered septicemia (N = 3) caused by biofilm implantation without antibiotics (sepsis) reached levels over 1.500 pg/mL. www.nature.com/scientificreports/ To identify bacterial species in the scaffolds, MALDI-TOF MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry) analysis was performed ( Supplementary Fig. S3), showing the predominance of P. aeruginosa in 90% of samples before and after 10 days of implantation, 10% of samples had E. faecalis and 10% had S. aureus. Also, several other bacterial genera that were not initially inoculated were found in colonized wounds, but they were specific for each specific wound, where 20% of samples had Kocuria rosea, 10% had Escherichia coli, 10% had Pseudarthrobacter sulfonivorans and 10% had Pseudomonas straminea, showing a total of seven bacterial species detected in biofilm-containing scaffolds from this study (data not shown).

Systemic and local inflammation process induced by local infection. After confirming a local
infection response provoked by the biofilm implantation in wounds, their effect over the systemic inflammation process was characterized (Fig. 7). For systemic inflammation analysis, blood and lymphoid organs were extracted. Increased size of the spleen was observed due to biofilm implantation (Fig. 7A), which was confirmed with a significant increase in its weight, compared to the control group (Fig. 7B). Lymph nodes and thymus did not show variations in size nor weight under biofilm infection (Fig. 7A,B). The microscopic inspection of spleen histological slides stained with H&E showed an increase in the ratio of lymph follicles/spleen area and the average amount of lymph follicles in the biofilm-infected groups (Fig. 7C).
To quantify systemic pro-inflammatory cytokines, blood was extracted from implanted animals, and plasma fraction was analyzed by flow cytometry (Fig. 7D). None of the cytokines measured showed significant variations between groups treated with ciprofloxacin (control or biofilm-infected groups). In contrast, cytokine levels from animals with biofilm-containing scaffolds that were not treated with antibiotics, and suffered septicemia, showed elevated levels for cytokines MCP-1 (monocyte chemoattractant protein-1), IL-10 and IL-6 compared to groups treated with ciprofloxacin and control (Suppl. Fig. S2).
Once the systemic inflammatory response was evaluated, the local inflammatory process was also studied (Fig. 7E,F). Here cytokines obtained from protein extracts of the implanted scaffolds were quantified, showing significantly higher levels of IFN-γ and IL-10 in infected samples compared to sterile ones (Fig. 7E). To evaluate leukocyte infiltration, histological sections from scaffolds were processed for immunohistochemical and histochemical analysis to detect macrophages and neutrophils (Fig. 7F). Results show that in the presence of biofilm www.nature.com/scientificreports/ there was a marked response from the host, given by the higher presence of macrophages and neutrophils in the scaffold.
To qualitatively assess the effect of biofilm in the regeneration process, paraffin sections were processed for Masson's trichrome stain and immunohistochemistry (Fig. 8). Results showed the presence of endothelial cells marked with cluster of differentiation 31 (CD31, Fig. 8A), myofibroblasts with alpha-smooth muscle actin (α-SMA, Fig. 8B) and cells under cell proliferation (ki67, Fig. 8C), consistent with the regeneration process in control animals implanted with sterile scaffolds. In the case of biofilm-infected groups, these markers were not visualized, while a high number of leucocyte infiltrate was observed.

Discussion
Despite decades of research, the role of biofilm-mediated infection in chronic wounds has not yet been completely elucidated, hence the development of in vitro and in vivo biofilm models to better understand their biology, as well as their role in the regeneration process, is required to develop novel strategies for chronic wound management. Thus, the aim of this work was to establish a biofilm-infected in vitro model, which can be further implanted in a wound model to evaluate the effect of biofilms in vivo.
Initially, conditions were established to grow biofilms in commercially available collagen-GAG based scaffolds, that resemble the skin extracellular matrix in terms of structure and composition, which has been broadly described in research and for its clinical use for dermal regeneration 39 . Using an MTT assay, where formazan crystals remain intracellularly 40 , viable bacteria within the biomaterial were localized and quantified 40 . Results confirmed the formation of a metabolically active biofilm formed on the biomaterial's surface, which, for low cell density biofilms, was homogeneously distributed, while those formed from high bacterial loads were at the center and bottom of the scaffold. This suggests the formation of metabolic gradients due to oxygen or nutrient's chemical gradients 11 , which can correlate with their ability to tolerate antibiotics 41 . CLSM and SEM analysis was consistent, demonstrating that biofilms organized in bacterial aggregates attached to the pores of the scaffold. Possibly, biofilm formation started with bacteria that attach to the surface and latter multiply, secreting EPS to become a thick-layer biofilm established in the scaffold, following a typical growth cycle 42,43 .
In the present in vitro model, tolerance against ciprofloxacin and gentamicin treatment was corroborated in biofilms formed over collagen-scaffolds, as previously reported for P. aeruginosa over gels (with 10 2 CFU/gel after 24 h) 30 and mixed-species biofilms in the Lubbock model that reaches approximately 10 5 to 10 6 CFU/gr of tissue after 5 h 44 . Our results showed a 100-and 20-fold increase in the MIC of ciprofloxacin and gentamicin for bacteria forming biofilms compared to planktonic, as described for chronic wound biofilms 45,46 , indicating that the proposed in vitro model is viable and functional. Moreover, in vivo results confirmed that ciprofloxacin therapy prevents septicemia, since bacterial inoculation without treatment resulted in 100% mortality of mice, an outcome previously reported in mice and horse models 36,38,47 . Despite the daily dose of ciprofloxacin 10 mg/ kg, biofilm implantation generated a persistent infection, with a detrimental effect over animal welfare as seen in higher health score, permanent weight loss and initial febrile state, resembling the severely affected life quality of chronic wound patients 5 . www.nature.com/scientificreports/ The model described here demonstrates that a low-density initial inoculum can form biofilms of 10 8 CFU/ mL after 24 h, and reach over 10 9 CFU/gr of tissue after ten days in vivo, resembling a more clinically relevant initial bacterial exposure to patients 48 . Similarly, CFU counting revealed groups of non-infected, mildly, or highly infected wounds, which correlated with gradients of infiltration and migration of PMN cells and macrophages visualized by histology. This observation mimics the categorization of patient's wounds into 'light' , 'occasional' , 'moderate' , or 'heavy growth' infected 49 . Regarding the bacterial diversity 50 despite Pseudomonas dominated the infection process in most animals, there was a less frequent presence of S. aureus, E. faecalis, and the occasional identification of seven bacterial species from mice and the environment, which suggests that the proposed scaffold-infected model has a heterogeneous polymicrobial and dynamic composition like chronic wounds 17 .
At a systemic level, an increased size of spleen and histological analysis shows that implantation of biofilmcontaining scaffolds induce splenomegaly, with increased size and number of lymph follicles where lymphocyte T and B differentiate and proliferate 52 , suggesting a high immunological activity of the spleen probably in response to bacterial lipopolysaccharides, as it has been previously reported 53 . In addition, levels of pro-calcitonin were significantly higher for biofilm-infected groups compared to controls, with protein concentration values in plasma within reported ranges for chronic-infected patients 54,55 , demonstrating that, besides detrimental effect over welfare, other systemic effects that mimics patient's symptoms were also present.
Locally, quantitation of cytokines from the implanted scaffolds showed an increase in IFN-γ and IL-10 cytokines, while pro-inflammatory cytokines IL-12, TNF-α, IL-6 and MCP-1 did not change. Interestingly, it has been reported that IL-10 can be enhanced by pathogens [56][57][58] through TLR activation in macrophages by S. aureus 59 , and P. aeruginosa 60 , inhibiting the production of IFN-γ, IL-12 and TNF-α 59 . Hence, biofilm-containing scaffolds may be inducing IL-10 as an evasion mechanism, perpetuating the local infective process. Regarding the morphology at the wound site, histologic analysis indicated two types of cellular interactions: at the wound edge, there was an influx of PMN cells as an inflammatory response against biofilm, whereas in the wound bed the low count of cells indicated inhibition of cellularization and epithelialization. Consistent with this observation, a qualitative histological analysis shows the lack of myofibroblasts, cells in a proliferative state and endothelial cells are linked to a reduced capacity for regeneration, suggesting that biofilm infection seriously impairs wound healing, as previously described in other animal models 34,35,38 .
In conclusion, this study provides a biofilm-infected wound model that resembles most clinical aspects of chronic wounds. The establishment of biofilms over a scaffold provides an implantable in vitro model, with tested antimicrobial tolerance and bacterial loads, which can be used for screening of therapies prior to implantation. In parallel, the in vivo wound model described here avoids wound contraction, resembling scaffold-based dermal regeneration in humans, reporting systemic and local effects of a polymicrobial biofilm infection over health and inflammation, similar to chronic wound patients' symptoms. The incorporation of guidelines regarding the welfare and health state of animals and pro-calcitonin quantitation, which have not been assessed in other animal models, represents another contribution of this in vitro and in vivo infected wound model, making it suitable for testing novel therapies for chronic wound management and treatment.

Materials and methods
Biofilm formation in scaffolds. Bacterial strains of P. aeruginosa (ATCC 27,853), S. aureus (ATCC 29,213) and E. faecalis (ATCC 29,212) were cultured overnight at 30 °C in Luria Bertani broth, diluted (1:3) and incubated 1.5-2 h until exponential growth. Then, cells were harvested, counted, and resuspended to equal species proportions to 10 2 or 10 8 cells/mL, and seeded in 12-mm diameter and 2-mm thickness collagen-glycosaminoglycan scaffolds (Integra ® DRT, kindly provided by Integra LifeSciences), which were previously dried with a sterile gauze. After 30 min of initial bacterial attachment, scaffolds were dried again, rehydrated with 1 mL M9 minimal culture media and incubated at 30 °C for 24 h under static conditions to allow biofilm formation. To avoid retention of planktonic cells, supernatants were removed and biofilm-containing scaffolds were dried with sterile gauzes and processed for further analysis or animal implantation.
MTT metabolic assays. Seeded or sterile scaffolds were incubated in 90 µL of M9 media containing 10 µL of 5 mg/mL MTT (Sigma Aldrich) for 4 h at 37 °C. Next, scaffolds were imaged using a stereomicroscope (Leica Biosystems). Afterwards, scaffolds were incubated in 500 mL dimethyl sulfoxide (Sigma Aldrich) until all formazan blue was dissolved. Absorbance of formazan blue was measured at 570 nm and 550 nm was used as reference 61 . Bacterial quantitation and species identification. For in vitro studies ("Biofilm formation in scaffolds" and "Antibiotic tolerance assay"), biofilm-containing scaffolds were dried with sterile gauzes and mechanically disrupted by pipetting in 0.5 mL PBS, 2 min vortex, serially diluted and seeded in Trypcase Soy agar + 5% sheep blood plates (Biomérieux). After 24 h of incubation at 30 °C, colony forming units (CFU) were quantified. For in vivo samples (Sect. 2.7), a quarter of each implanted scaffold was weighed, resuspended in 0.5 mL phosphate buffered saline (PBS) vortexed for 2 min, serially diluted and seeded for further CFU counting. For species determination, isolated colonies were identified by MALDI-TOF Mass Spectrometry (Bruker Daltonik) as previously published 62  Confocal laser scanning microcopy and image processing. Scaffolds from in vitro studies ("Biofilm formation in scaffolds" and "Antibiotic tolerance assay"), were dried using sterile gauzes, fixed with 0.5 mL of 4% paraformaldehyde in PBS for 1 h at room temperature, dried with gauze and washed three times with distilled  65 , and the scaffolds were subjected to CLSM analysis following the protocol described above ("Confocal laser scanning microcopy (CLSM) and image processing"). Sterile scaffolds with M9 media were used as blank control.
Scaffold implantation procedure. Surgeries were performed as described before, with slight modifications 66 . Briefly, 7-9 weeks old (19 to 25 g body weight) female C57/BL6 mice were anesthetized with ketamine (87.5 mg/kg) and xylazine (9 mg/kg), and hair was removed using a pet clipper and shaving cream (Veet®, Reckitt Benckiser). To prevent eye damage during surgery, an ophthalmic gel was applied (Nicotears®, Saval Laboratories), and 0.5 mL of saline solution was subcutaneously injected to avoid dehydration 66 . Afterwards, under biosafety cabinet, skin was sterilized with 2% chlorhexidine gluconate (Difem® Laboratories) and two 10-mm diameter bilateral full skin defects were surgically created in the back of the animal, using fine surgical scissors. Further, a titanized mesh of 13-mm diameter (TiMesh™, Pfm medical) was placed under the wound edges, and scaffolds were fixed by six single knots using non-absorbable surgical sutures (Ethilon 5/0, Johnson and Johnson). Finally, the implanted wounds were covered with a transparent dressing (Tegaderm™, 3 M). The day before surgery ciprofloxacin (30 mg/kg) was given as prophylaxis and injected during nine days as antibiotic therapy. For analgesic and antipyretic treatment, meloxicam (5 mg/kg) was administered before surgery and daily, after three days post-surgery. All animal experiments were performed according to protocols approved by the Ethics Committee of Universidad de Chile (19237-INT-UCH), and were conducted in accordance with the current Chilean legislation, the 3Rs guidelines from the UK National Centre for the Replacement Refinement and Reduction of Animals in Research, and the guidelines of the Care and Use of Laboratory Animals published by the US National Institute of Health. The study is reported according to the ARRIVE guideline 2.0.
General health assessment. Animal health was daily supervised, following a general health score assessment sheet used for surgical interventions, that is based on Grimace Scale and NC3Rs guidelines 67 . This assigns 0 to 3 points for five parameters: (a) Weight loss ranging from < 5, 10, 20% of initial body weight; (b) General aspect of hair, posture, and secretions from eyes or ears; (c) Wound aspect, ranging from adequate hemostasia without edema to bleeding, inflammation or infection (yellow secretions); (d) Spontaneous behavior within the cage, from normal to diminished mobility and even self-mutilations; (e) Behavior in response to manipulation, from normal resistance to aggression or weakness with signs of pain. When two or more parameters have a score of 3, they increase to 4. Fifteen points was the humanitarian endpoint of experiment in case of septicemia, using an overdose of ketamine/xylazine intraperitoneal as euthanasia. Body temperature was daily measured with an infrared thermometer at the shaved back of the mice.
Euthanasia and sample collection. After ten days of scaffold implantation, animals were euthanized by intraperitoneal overdose of ketamine/xylazine. Intracardiac blood was collected, clotted on ice for 1 h, centrifuged at 2000 g for 10 min at 4 °C, and 200 µL of blood serum was stored at − 80 °C for further analysis 68 . The remaining blood sample was resuspended in 0.5 mL PBS for CFU determination. Scaffolds were harvested and divided into quarters for protein extraction (stored at -80 °C), histology (fixed in 4% formaldehyde for 24 h) and CFU counting (resuspended in 0.5 mL sterile PBS). Additionally, for histological analysis, a sample of the skin from the wound edge was also obtained and fixed in 4% formaldehyde for 24 h. Thymus, spleen, and lymph nodes were harvested, weighed, imaged using a stereomicroscope (Leica Biosystems), and fixed for histology.